Photoexcitation of Tryptophan Groups Induces Reduction of Two Disulfide Bonds in Goat α-Lactalbumin

Abstract

Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

“…It has been demonstrated that electron attachment to a disulfide bond leads to significant bond weakening and formation of stable radical species at low temperature in solution [31,32]. The reduction of disulfide bonds by photoexcitation of tryptophan groups in goat ␣-lactalbumin has been described before [33]. This process involves transfer of photo-electrons from a proximal tryptophan to the disulfide bond and subsequent cleavage into a thiolate ion and thiyl radical (see Supplemental Materials).…”

Effect of pH and Light on Aggregation and Conformation of an IgG1 mAb

“…1 An important feature of tryptophan photolysis is the potential for photoinduced electron transfer to nearby disulfide bridges 1 where disulfide reduction leads to a thiolate and a thiyl radical. 1 Thiyl radicals can form also via direct light-induced homolysis of cystine disulfide bonds [29][30][31][32] and a number of reaction products have been characterized for thiyl radical pairs generated in such way 1, [33][34][35][36][37] For completeness, we note that disulfide bonds can also cleave via C-S bond breakage into carbon-centered radicals and perthiyl radicals, but this process appears to prevail for β-alkyl substituted disulfides such as, for example, penicillamine. 30 For comparison, the light-induced cleavage of glutathione disulfide led predominantly to thiyl radicals.…”

A New Ligation Strategy for Peptide and Protein Glycosylation: Photoinduced Thiol–Ene Coupling

“…[32] From a mechanistic point of view, it can be speculated that tryptophan residues present in BSA were responsible for the observed hyperglycosylation. It has been reported that prolonged near-UV irradiation of goat a-lactalbumin [33] or hen egg white lysozyme [34] induces tryptophan residue excitation and this is followed by electron transfer to some nearby cystine S À S bonds with consequential cleavage into thiol radical and thiolate anion. The same mechanism may be operative in the BSA irradiation, thus providing an extra number of thiol radicals centered on the former cystine residues.…”